The present invention relates to a diffusion promoting device connected to just before a separation column and having a function for improving detection sensitivity in a low flow velocity gradient high performance liquid chromatography system, to a method for improving detection sensitivity of a target component by connecting the diffusion promoting device, and to an analytical method using the system.
High performance liquid chromatography has been frequently used for analyzing a trace amount of components in a sample. In recent years, high performance liquid chromatography is used in a system for simultaneously separating and identifying the component in a high sensitivity by combining with a mass spectrometer or a nuclear magnetic resonance spectrometer. For example, JP-A 3-175355 discloses an apparatus and a method for conversion of mobile phases in a high performance liquid chromatography-mass spectrometer system, and an apparatus for trapping a target components in a trapping column.
However, the permissible flow velocity of the mobile phase capable of flowing into the mass spectrometer is several tens microliters/min in the high performance liquid chromatography-mass spectrometer system from the view point of analytical accuracy of the mass spectrometer. A tube having a smaller inner diameter than conventional tubes is often used in the low flow velocity high performance liquid chromatography system as described above, in order to suppress diffusion of the target component in the tube of a liquid flow line. The inner diameter of the tube is generally determined by considering the inner diameter of a separation column and linear velocity in the tube.
When a tube having an inner diameter of 0.1 mm or less is used in the low flow velocity high performance liquid chromatography system, however, the tube may be blocked with fine particles to make an analysis by low flow velocity high performance liquid chromatography difficult.
It is known in the art, on the other hand, that an effect for concentrating the target component at the top portion of the separation column may be obtained when a gradient elution method is used in low flow velocity high performance liquid chromatography, that is in low flow velocity gradient high performance liquid chromatography. For example, JP-A 3-175355 discloses an example in which a tube having a larger inner diameter than usual, i.e. 0.8 mm, and a length of 100 mm is connected at just before the separation column for efficiently obtaining the concentration effect. It reports that a gradient effect is obtained in the tube and peak resolution is improved by using such column as described above. However, since a tube as long as 100 mm is use d in the method described above, it may be conjectured that improvement of resolution is not always realized depending o n the kind of the separation column used, and on analysis conditions in high performance liquid chromatography.
Accordingly, development of an apparatus capable of enhancing analytical and quantitative sensitivity is urgently desired in the low flow velocity gradient high performance liquid chromatographic apparatus for analysis and assessment of a trace amount of biological components, and impurities in environmental samples and pharmaceuticals.